Should siRNA sequence be 100% complementary? - (Apr/02/2008 )
I'm not from the field and I want to silence a gene in PC12 cells. I got the sequence from a collaborator and it was made for silencing the gene in HEK-293 cells. I checked the sequence and there's one base pair different.
I was told that siRNAs are much more developed for human-derived cells, but I saw some papers with PC12 cells (rat). What do you think of it? Do I have good chances to be successful? I don't know if I should put money and time on this...
The sequence works fine in HEK-293 cells.
The one thing I am sure about, is that the more homology the siRNA has with the sequence, the more effective your knockdown will be. So if you are to tranfect or electroporate the siRNA into your cells, I would recomment that you order anotre siRNA with 100% homology. But if the siRNA is to be put into a viral vector in a shRNA, the amount of work is far greater. In that case, I would try with the human sequence and monitor the knockdown with qPCR and western blot.
Hope that helps
It also depends on where the mismatch is. If the mismatch is in the seed sequence of the guide strand, it may not work at all. I would suggest that you design a new siRNA against the rat sequence.
That don't HAVE to be exactly complementary to get good knockdown. A single base difference won't hamper it too much if it's in the middle of the siRNA sequence (Remember, miRNAs work in many cases as imperfect complements). But if you're going to be doing multiple exps with this stuff you really should make it yourself as a perfect complement. Then you don't have to explain it away in your methods when you publish a paper with it
Hey, thanks a lot for your opinions... guess it's not a good idea to spend a lot of time in this if it's not perfectly complementary!
My problem is that I would have to transfect two proteins plus the siRNA in the HEK-293 cells, that's why I wanted to use the PC12.
I don't know if it'll be possible for me to order a sequence, let's see!